Assay for adrenal autoantigen

- RSR Limited

Adrenal autoantigen is isolated and characterised as a protein obtainable from the microsome fraction of the adrenal gland, having a molecular weight of about 55,000 and antigenic towards adrenal autoantibody. The protein is used in a method for detecting adrenal autoantibodies.

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Claims

1. A method for diagnosing Addison's disease in a patient, comprising:

obtaining a sample containing antibodies from the patient; and
detecting, in the sample, the presence or absence of antibodies which specifically bind a substantially pure microsomal protein comprising an epitope for adrenal autoantibody, having an observed molecular weight of approximately 55,000 as measured by gel electrophoresis and obtained by:
homogenizing mammalian adrenal glands,
subjecting the homogenate to differential centrifugation to obtain a microsome fraction,
suspending the microsome fraction in a phosphate buffer,
centrifuging the suspension in the presence of sodium cholate to form a supernatant,
adding polyethylene glycol and further sodium cholate to the supernatant and mixing the supematant,
centrifuging the thus mixed supernatant to form a pellet,
resuspending the pellet in aqueous sodium cholate to form a suspension,
dialyzing the suspension obtained by resuspending the pellet against aqueous sodium cholate to form a solubilized microsome preparation, and
purifying the solubilized microsome preparation by column chromatography on a C.sub.8 reversed phase column equilibrated with 50 mM phosphate buffer, pH 7.0, 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT, and eluted with increasing concentration of detergent to obtain a column fraction corresponding to peak VI of FIG. 1 containing the microsomal protein;
wherein presence of said antibodies in said sample is indicative of Addison's disease in the patient.
Referenced Cited
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Foreign Patent Documents
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Other references
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Patent History
Patent number: 5705400
Type: Grant
Filed: Sep 2, 1993
Date of Patent: Jan 6, 1998
Assignee: RSR Limited (Cardiff)
Inventor: Jadwiga Maria Furmaniak-Wehr (Cardiff)
Primary Examiner: Paula K. Hutzell
Assistant Examiner: James L. Grun
Law Firm: Baker & Botts, L.L.P.
Application Number: 8/115,052